Atherosclerosis and hypertension in relation to some trace elements in tissues-1.

World Review of Nutrition and Dietetics, vol. 20, pp. 299-326 (Karger, Basel 1975)

Atherosclerosis and Hypertension in Relation to Some Trace Elements in Tissues1 Α. W. Vooii.s, M. S. SHUØN and P. N. GALLAGHER Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, N.C.

Contents 300 301 303 303 304 305 306 306 307 307 307 308 308 316 318 318 319

1 We thank Mr. G. WOODWARD for technical assistance. This investigation was supported by contract No. CPA 70-105 from the Environmental Protection Agency. This paper does not necessarily represent the views or stated policy of this agency.

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I. Description of the Problem A. Cadmium Effects B. Zinc Effects C. Effect of Element Interactions D. Geographical Coincidence II. Materials and Methods A. Pathological Rating B. Preconcentration C. Manipulation of Ash D. Chemical Analysis E. Precision of Analytical Techniques F. Statistical Analysis III. Results IV. Interpretation of Results V. Conclusion Summary References

VGGRS/SHUMAN/GALLAGHER

300

Cardiovascular disease mortality of nonmetropolitan residents in the US has a peculiar geographical pattern [SAUER, 1962; SAUER and BRAND, 1968, 1971]. Migration studies [SAUER, 1962, 1967] have validated the concept of geographical disease determinants in this context. Moreover, a growing body of evidence indicates that the well-known [CRAwFοmι et al., 1971] negative association between hardness of drinking water and cardiovascular death may be due to environmental influence upon the host organism. However, there is still uncertainty about the biological nature of this environmental influence, although a number of hypotheses has been postulated [as reviewed, for instance, by MASIRoNI, 1969]. In contrast to the relative invariability of human and animal tissue levels of the bulk elements and trace elements controlled by a homoiostatic host mechanism, there is marked geographical [TIPToi et al., 1965] and individual [WESTER, 1965] variation in the tissue concentration of those trace elements which appear to be lacking such homoiostatic mechanisms in the host. It is the latter elements that seem currently of most interest in this respect, because only they permit causal explanations through their differential presence in the relevant organ tissues. Three theories about the possible nature of the environmental determinant are emerging. The first theory is that this determinant of cardiovascular disease is a deficient intake of micronutrients. SHACKLETTE et al. [1970] have concluded from extensive soil and plant analyses in Georgia covering many trace elements that if geochemical differences do indeed have a causal relationship to death from cardiovascular disease, the cause would appear to be a deficiency rather than an excess of one or more of the elements studied. The second theory on the nature of the cardiovascular disease determinant is an excess of trace substances. Cadmium has been incriminated in relatively recent literature (see below). The third theory postulates competitive inhibition among elements which have certain chemical characteristics in common as do, for instance, cadmium, copper and zinc [HILL and ΜΑτRονΕ, 1970] (see below). This third theory could encompass to some extent the two previously mentioned. Among the trace elements investigated, mainly cadmium and zinc have conceptual relevance to cardiovascular disease. In the next section, the literature with respect to these two elements and their interaction is briefly reviewed. This review will be followed by some notes on the geographical coincidence of excessive cardiovascular disease occurring together with altered micronutrient intake as indexed by environmental characteristics.


I. Description of the Problem

Atherosclerosis and Hypertension

301

Reviews of cadmium effects are given by FLICK et al. [1971] and in more detail by FRIBERG et al. [1971]. Autopsies on cadmium-exposed industrial workers have revealed specific lesions which generally were confined to the proximal tubules of the kidney [FRIBERG et al., 1971, pp. 6-11]. Only in Japan and perhaps in France more developed cases of Fanconi syndrome have been recorded [FRIBERG et al., 1971; ΚΟΒλΥΑSΗτ, 1971]. AXELSSON et al. [1968] have described histologic changes in the proximal tubules of cadmium-poisoned rabbits. More recently, Nismzuii [1972] gave a similar description for the rat. Mice injected with water-soluble radioactive cadmium showed microradiographic cadmium localization in the proximal tubules of the kidney [BERLIN et al., 1964]. High dosage of cadmium (for instance, 5 ppm in drinking water consumed by rats for more than 15 months) resulted in hypertension [KANIsAWA and SCHROEDER, 1969]. In dogs, such cadmium administration had a direct enhancing effect on the renal tubular sodium reabsorption [VANDER, 1962 a]. The site of this cadmium action has been located through stop-flow studies. It was found that the cadmium increases the proximal tubular sodium reabsorption [VANDER, 1962 b]. Low-dosed cadmium administration resulted in a decrease of the renal sodium excretion [PERRY et al., 1971]. Such a dosage could produce labile systolic hypertension in female rats [SCHROEDER and luNTh , 1962], but other investigators as reviewed by FRIBERG et al. [1971, pp. 6-33] could not replicate this finding. In male rats, SCHROEDER and VliTON [1962] and SCHROEDER et al. [1970] were able to produce hypertension when they added laC1 to the diet. Likewise, LΕNER [as quoted by FRIBERG et al., 1971, pp. 6-33] could produce an increase in the blood pressure of rats by low-dosed cadmium after pretreatment with 2 % NaCI in the drinking water for 50 days. Moreover, low-dosed cadmium produced a decrease in peripheral vascular response to noradrenalin or angiotensin [PERRY et al., 1971 ; THuND et al., 1970; VOUKILIS et al., 1969 ; SCHROEDER et al., 1970]. Since such a response may be part of a feedback mechanism regulating plasma renin activity, it is of interest that inability to produce average amounts of renin may preclude essential hypertension [BRuINER et al., 1972]. Conversely, rats kept lifelong on an unusually low-cadmium diet had blood pressures below normal [KANIsAWA and SCHROEDER, 1969]. This find-


A. Cadmium Effects

302

ing is of interest for 2 reasons : (1) the `normal' blood pressure of US residents over 50 years of age may not be optimal [HENRY and CASSEL, 1969], and (2) conceivably the restriction of NaCl intake could reverse the upward age trend of blood pressure [BORST et al., 1963; DAHL, 1972], since such a trend may prevail only in the industrialized areas [see the data presented by HENRY and CASSEL, 1969, and by ΕΠSrrnν and ECKOFF, 1967], where cadmium levels in the kidney are highest. This notion is supported by recent findings suggesting that renin secretion is necessary in the causation of essential hypertension [BRUINER et al., 1972]. An increase in aortic lipids and in the production of aortic lipid plaques and sclerosis in small arteries of heart, liver and other organs was observed by SCHROEDER et al. [1965 a, 1965 b], and KANIsAWA and SCHROEDER [1969] after administration of cadmium to mice. They concluded that such changes were secondary to the induced hypertension. Experimental hypertension induced in cholesterolized rabbits consistently resulted in acceleration and aggravation of the cholesterol-induced atherosclerosis [Awlrscκκοω, 1933; NuzuI et al., 1926; DILL and ISENHouR, 1942; WILENS, 1943]. It would seem that the cholesterolized rabbit is a better experimental model for simulating cadmium effects in the industrialized overfed and under-exercized atheromaprone human, than is the atheroma-resistant mouse or rat. So much for the experimental evidence that cadmium may be a determinant of cardiovascular disease. Epidemiological observations in this respect include the following. Cadmium is known to accumulate with age in the human kidney cortex, where it reaches levels at least 10 times those in other tissues [TIPTON et al., 1963, Ττ roν and Cooκ, 1963]. The mean and median concentrations of cadmium in the renal cortex of autopsy populations in various nations cover a large range. Some technically developed nations have average levels 8 times higher than the lowest among the underdeveloped nations [PERRY et al., 1961]. Nation for nation, these averages are positively associated with mortality due to hypertensive disease [SCHROEDER, 1965]. Experimentally elevated concentrations of cadmium in the kidneys of mice and rats, whose lifetimes are thereby decreased, are 3- to 4-fold lower than those found normally in the kidneys of autopsied US residents [SCHROEDER et al., 1963 a, b]. A positive association between hypertensive disease and cadmium levels in the renal tissue of individuals has also been reported [SCHROEDER, 1964, 1965; LEVER and BIBR, 1971], but not consistently [MoRGAI, 1969; NoGAWA and KAwAio, 1969; HICKEY et al., 1967]. Within the US, cadmium levels in the air were found to be associated with mortality


VGORS/SHUMAN/GALLAGHER


303

from heart disease excluding rheumatic heart disease [CARROLL, 1966], but negative results have also been reported [HuiT et al., 1970].

B. Zinc Effects For reviews of zinc effects, see Fox [1970] and SCHROEDER et al. [1967 b]. The presence of zinc is required for the functioning of a number of enzymes involved in protein and nucleic acid metabolism and collagen synthesis [SANDSTEAD et al., 1970; SANDSTEAD and TERHUNE, 1971]. There is very minimal storage of readily available zinc in the growing male rat [PETERING et al., 1971] and in young calves and lambs [MILLS et al., 1967]. In humans the levels of zinc in various organs decrease with age [SCHROEDER et al., 1967 b]. They are lower in industrialized than in the developing countries [Tιrrον et al., 1965], and may be decreasing by the advancement of food refining practices [SCHROEDER, 1971]. Zinc deficiency has been associated with impaired wound healing [GRεAΝEs and SKILLEN, 1970; SERJEANT et al., 1970] and with defective tissue repair in general [OBERLEAS et al., 1971; PORIES et al., 1967; WESTMORELAND, 1971; BERGMAN, 1970; MESROBIAN, 1971 ; PORIES, 1971 ; HENZEL et al., 1967]. In addition, HEIZEL [1971] and HEIZEL et al. [1969] postulated that various atherosclerotic diseases can be treated with zinc resulting in repair towards the nondiseased state. ISAACS et al. [1971] likewise treated such patients with zinc (and other trace substances) with favorable results.

Α potentially useful notion in trace element epidemiology is the postulate of competitive inhibition among elements with certain similar chemical attributes if one of them normally functions in an enzyme complex. HILL and MATRONS [1970] have suggested that these attributes are likely to include the angular distribution of electronic orbitals of the atoms, and cited zinc and cadmium as one of their examples. It has been shown by others as well [PARIZEK, 1957 ; PARIZEK et al., 1971; MASON and YOUNG, 1967] that the noxious effects of cadmium can be decreased by the simultaneous administration of zinc. For these and similar reasons SCHROEDER et al. [1967b] proposed that the cadmium-zinc ratio of kidney concentrations is a meaningful index of the


C. Effect of Element Interactions


304

trace metal imbalance suspected to be a determinant of cardiovascular disease. He tested this idea by treating cadmium-poisoned rats with a chelate and zinc, and could demonstrate not only a decrease in the cadmium-zinc ratio, but also a reversal of the hypertension [SCHROEDER et al., 1967 a], as well as a reversal of the decrease in vascular reactivity [VOUKILIS et al., 1969]. The postulate of the inhibition of zinc-dependent enzymes by cadmium permits a new tentative explanation of the mechanism of cadmium hypertension. Some of the body chemoreceptors, namely the taste buds, need zinc in order to function [HENKIN et al., 1971]. The function of other chemoreceptors, namely those in the smell organ, is impaired by cadmium [FRIBERG, 1950; BAADER, 1951]. Thus, the observed association between hypertension and decreased sensitivity of taste for NaCl [FALL'S et al., 1962] suggests a new interpretation. Current opinion [DAVIS, 1971 ; SΤΑτντευ, 1963] favors the hypothesis of sodium perception by the juxtaglomerular cells in the regulation of renin secretion. This implies the possibility that sodium chemoreception here may be impaired by relative zinc deficiency, resulting in hyponatruria as an early sign of cadmium intoxication [PERRY et al., 1971]. There are other mechanisms by which the noxious effects of cadmium may be decreased through element interaction. The presence of calcium [SCHROEDER et al., 1967a] and selenium [PARIZEK, 1957; PARIzEK et al., 1971; MASON and YouNG, 1967] have been shown to protect against cadmium toxicity. Finally, the presence of basic calcium salts in the drinking water could decrease its uptake of cadmium from galvanized pipes [SCHROEDER et al., 1967b].

Interest in the negative association between water hardness and cardiovascular death was initiated by KOBAYASHI'S finding [1957] that stroke mortality in Japan is associated with acidity of the river water. It is again in Japan that relatively high cadmium-zinc ratios in the kidney tissues have been found, which were tentatively linked to cardiovascular mortality [PERRY et al., 1961]. All nonsaline water in Japan seems to be soft. In the US, MORTON [1971] has commented on the remarkable contiguity of the regions with relative excess of hypertensive heart disease mortality and of the regions with relative deficit. The South Atlantic Coastal Plain is a predominantly rural area with a fairly stable population having an excess of stroke, hypertensive heart dis-


D. Geographical Coincidence


305

ease and atherosclerotic heart disease mortality [SAUER and BRAND, 1968, 1971; ΗΕΥΜΑΝ et al., 1971]. Furthermore, since disturbance of the blood pressure regulation may be a risk factor of sudden death in patients with compromised coronary circulation, it is noteworthy that in the South Atlantic Coastal Plain the rate of sudden heart death also seems to be excessive [VooRs, 1971]. Because this area has supplies of soft water and a leached soil where zinc, selenium, manganese, copper, cobalt and phosphor deficiencies have been reported [SCHUETTE, 1964], some relative nutrient deficiency could play a role in the hypertension excess of this region. Particularly the geographical coincidence between excessive mortality due to hypertensive heart disease and stroke [SAUER and BRAND, 1968, 1971] and zinc deficiency in the soil [THoMPsoN, 1957] is impressive when this region is compared with the rest of the US.

The trace element survey of selected organs in a geographically defined autopsy population is a suitable research method for the present problem of testing biological hypotheses based on ecological associations. This method has been applied first by Τι τoν [1960], Τ ΠΤ0Ν and Cook [1963] and Τι τοi et al. [1963, 1965]. Potentially this technique can answer the question whether trace element levels in the relevant human organ, when assessed at the time at which the pathogenesis occurs, is associated with the occurrence of the disease. Because of this temporal restriction, the question can be answered reliably only for toxic elements which accumulate in the body organs. In order to gain more insight in this problem, an autopsy survey of a few trace elements was applied to a population of North Carolina residents, part of whom lived in the Coastal Plain, a rural area with high death rates due to atherosclerotic heart disease, stroke and hypertensive heart disease. The area has soft water and leached soils. The remaining part of the autopsy subjects lived mainly in the Piedmont, an industrialized area with less soil leaching and only moderate cardiovascular mortality, taking its degree of urbanization into account. 106 autopsies occurring during the first half of the year 1971 on persons aged 16-91 were contributed by 7 hospitals in central and eastern North Carolina. The hospital population represented by the study sample had an autopsy rate well over 60 %. Although there was little selection exercised in the acceptance of autopsies into the study, some preference was given to


IL Materials and Methods


306

white males who had died of cardiovascular disease. Up to 10 g of tissue from the following 6 organs were collected, deep-frozen and trimmed by one of us (P.N.G.) under precautions against trace element contamination. Organs were selected for relevance to trace metal homoiostasis. They were: aorta, bone (rib), kidney (cortex), liver, lung and skin (abdominal). Pathological sites in the organs were avoided where possible. Age, race and sex were obtained from hospital records. Usual residence, smoking habits and occupational exposure were obtained via a short questionnaire sent to relatives of the decedents. The section of this questionnaire relevant to the present study is repioduced in the appendix.

A. Pathological Ratings Hypertension was indexed by the cardiac weight under exclusion of valvular disease [NETSKY et al., 1969]. Atherosclerosis status was assessed from the autopsy report as follows. Atherosclerosis was rated `positive' under any one of 3 conditions : (1) death was attributed to stroke or ischemic heart disease; (2) atherosclerosis was listed in the pathological diagnosis, and (3) the pathological description pointed to a degree of atherosclerosis which was excessive compared with the realistic biological optimum for the age in question. Cancer status was also assessed from the autopsy report. Any autopsy finding of clear-cut malignancy was rated as `cancer' except accidental findings of benign or in situ neoplasms. The latter were judged not to have exerted influence on the trace metal metabolism in general. On each autopsy summary paper used, the identification was made illegible. These summaries were stacked in random sequence and rated by an investigator (A.W.V.) After this, the same summaries were again 'shufed' in a new random order and rated again by the same investigator. All discordant ratings thus obtained were marked `indefinite' and only the concordant ratings were used in computations.

Wet samples of 2-4 g were weighed and freeze-dried for 8-12 h. Tissue samples were cut up and freeze-dried, and bone samples were then pulverized. The freeze-dried samples were weighed and placed in an International Plasma


Β. Preconcentration


307

Corporation Model 1101 plasma machine. Ashing conditions were typically 300 W of forward power (50 W/chamber) with 300 ml/min oxygen flow. Ashing time was 8-16 h, the longer times being necessary for bone samples. The ashed samples were removed from the asher, cooled, weighed and transferred to plastic-capped glass vials. C. Manipulation of Ash The sample ash was weighed to give a final analyte solution that was 2 mg of ash/ml solution. Samples with sufficient ash were made up to 5.0 ml. Samples were dissolved with glass-distilled water or with 1 : 2 HCl (Baker Ultrex) in the case of bone ash. A 1 : 10 dilution of this solution was made for the analysis of the metals Cd, Cu, Fe and Zn. D. Chemical Analysis The metals cadmium, copper, lead, iron and zinc were analyzed by atomic absorption spectroscopy using direct aspiration of the dissolved ash samples. Analysis was made using calibration curves. Analyses by standard addition performed for each metal revealed that within the precision of the analysis, no matrix interferences were present. Anodic stripping voltametry was used on several samples as an independent method for lead. This technique was valid for lead and these solutions if the solutions were made 0.3 to 0.5 i in HCI. Acidification was necessary to avoid complexation with the very minute amounts of soluble organic materials present in the solution of ash.

Precision of the atomic absorption and anodic stripping techniques was assessed by analyzing replicate solutions. Precision in the case of atomic absorption was a function of the absorption value (concentration) and of the background noise. Typical absorption values (these varied somewhat due to nonconstant noise levels) for the relative standard deviation were, for the high and low range of concentration, respectively: Cd, 5-25 %; Cu, 5-30 %; Fe, 1-10 %; Pb, 10-50 %; Zn, 1-10 %. Lead analysis in the case of anodic stripping analysis, gave a relative standard deviation of 7 % independent of concentration where electrolysis was long enough to give at least 1 ΜΑ of reverse current.


E. Precision of Analytical Techniques

VOORS/SHUMAN/GALLAGHER

3()

F. Statistical Analysis Differences in mean tissue metal levels between groups were tested by modified stepwise regression procedure, i. e. before and after linearly adjusting for age, race and sex. To this end a computer program was used, performing multiple regression analysis with dummy variables. The F-test was applied at the 5-percent level of significance. The effect of cardiac weight on the trace metal levels or ratios was likewise tested by multiple regression technique. Although the number of possible combinations of trace metal levels or ratios and organs was large, this study was from the onset focussed on cardiovascular disease and existing theories of its relationship with trace metals. For this reason the 5-percent level of significance was deemed appropriate. Element concentrations falling below the sensitivity threshold of the spectrometry method used were arbitrarily assigned three-fifths of the threshold value. III. Results Completed responses to the residence-and-exposure questionnaire were received for 64 % of the autopsies (67 % for the Piedmont residents, 58 % of

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HIGHLANDS

309


Table I. Mean ager, by race, sex, area, and atherosclerosis status Race-sex

Area

Atherosclerosis status

Piedmont Coastal other total Plain

White male Atherosclerotic Nonatherosclerotic Unknown

62.6 (18) 41.8 (4) 54.0 (2)

61.0 (5) 49.5 (2)

67.2 (6) 21.0 (3) 54.0 (3)

612 (29) 36.6 (9) 54.0 (5)

75.0 (1) 49.0 (1)

68.8 (13) 43.3 (6) 65.0 (1)

-

White female Atherosclerotic Nonatherosclerotic Unknown

66.2 (9) 47.0 (3)

-

74.7 (3) 35.0 (2) 65.0 (1)

Black male Atherosclerotic Nonatherosclerotic Unknown

50.0 (4) 50.2 (6) 34.0 (1)

55.3 (6) 39.3 (6) 59.0 (2)

-

43.0 (1) 70.0 (1)

53.2 (10) 44.6 (13) 55.5 (4)

Black female Atherosclerotic Nonatherosclerotic Unknown

57.7 (3) 45.0 (3) 41.0 (1)

47.5 (2) 33.2 (4)

-

17.0 (1) 67.0 (1) 30.0 (1)

47.5 (6) 41.9 (8) 35.5 (2)

Total Atherosclerotic Nonatherosclerotic Unknown

61.6 (34) 46.5 (16) 45.8 (4)

59.8 (16) 38.4 (14) 61.0 (3)

61.9 (8) 37.0 (6) 52.4 (5)

61.1 (58) 41.8 (36) 52.3 (12)

-

the Coastal Plain residents and 68 % for the others). For nonrespondents, usual place of residence was obtained from the death certificates. The case distribution over North Carolina counties of residence is mapped in figure 1. Most cases resided either in the Piedmont or in the Coastal Plain area. A breakdown of the population mean age by race, sex and atherosclerosis status for these two areas is given in table I. Atherosclerotic patients and whites tended to be older. Otherwise, no obvious differences were apparent. The analysis of the questionnaire responses indicated that the history of cigarette smoking, giving a quantitative estimate of daily cigarette smoking, was positively associated with the kidney cadmium level, accounting for


1 Sample size in brackets.

310

VOGRS/$HUMAN/GALLAGHER

Table II. Means element levele (ín ppm ash), by organ and by area Organ

Element

Área

cadmium

Aorta Piedmont Coastal Plain

40 37

copper

iron

lead

zinc

177 265

2,140 1,700

89 98

1,150 1,050

28 30

373 343

60 66

124 121

2,350 1,830

222 214

5,500 5,450

38 62

3,2783. 4 2,7703. 4

Piedmont Coastal Plain

269 176

377 382

14,800 14,100

81 111

4,200 3,740

Lung Piedmont Coastal Plain

77 75

324 138

14,100 12,400

34 41

1,050 1,040

57 32

113 88

1,300 1,240

37 24

581 548

Bone Piedmont Coastal Plain Kidney Piedmont Coastal Plain

5.6 5.1

Liver

Skin Piedmont Coastal Plain

28 % of its variation before, and for 29 % after adjusting for age. Reported exposure to fumes did not make a significant contribution, except in a few cases which remain to be investigated. Mean levels of each element in each organ are presented by region in table II. Cadmium, iron and zinc levels were slightly higher in the Piedmont


1 Although all tests were run adjusting for age, race and sex, the means shown were adjusted only if at least one of these three had an effect significant at the 5-percent level. 2 Sample sizes ranged at 50-53 for Piedmont and 30-33 for Coastal Plain, except for lung tissue (with sample sizes 46 and 29), for copper in bone (with sample sizes 21 and 7), and for iron in bone (with sample sizes 40 and 23). Decreased sample sizes are due to unavailability of specimens or to technical difficulties in the chemical analysis. 3 Difference between unadjusted means is significant at the 5-percent level. 4 Difference between means adjusted for age, race and sex is significant at the 5-percent level.


311

Table III. Means element levele (in ppm ash), by organ and by atherosclerosis status Organ Atherosclerosis status Aorta Atherosclerotic Nonatherosclerotic

Element cadmium

43 39

copper iron

lead

zinc

151 297

1,8803 2,5903

88 91

1,0403 1,3103

29 30

3753 3253

60 66

1263 1193

Bone Atherosclerotic Nonatherosclerotic Kidney Atherosclerotic Nonatherosclerotic

5.7 4.9 3,180 1,790

281 211

5,040 6,440

44 53

3,210 3,090

Atherosclerotic Nonatherosclerotic

264 176

472 386

13,900 15,800

68 114

4,100 4,240

Lung Atherosclerotic Nonatherosclerotic

91 53

310 135

14,200 14,000

29 39

1,070 1,023

52 36

90 143

1,320 1,460

38 17

570 615

Liver

Skin Atherosclerotic Nonatherosclerotic

area than in the Coastal Plan, and lead levels slightly lower. These differences were only in one instance (kidney zinc) statistically significant, but they were fairly consistent for the various organs. Lung and skin levels of most elements examined were higher in the Piedmont than in the Coastal Plain area, but none of these differences was statistically significant.


1 Although all tests were run adjusting for age, race and sex, the means shown were adjusted only if at least one of these three had an effect significant at the 5-percent level. 2 Sample sizes ranged at 54-62 for atherosclerotics and 39-43 for nonatheroscierotics except for lung tissue (with sample sizes 45-48 and 30-32), for copper in bone (with sample sizes 23 and 10), and for iron in bone (with sample sizes 43 and 35). Decreased samples sizes are due to unavailability of specimens or to technical difficulties in the chemical analysis. 3 Difference between unadjusted means is significant at the 5-percent level.

312

VOORS/SHUMAN/GALLAGRER

Table IV. Means cadmium and zinc levele (in ppm ash), by organ and by atherosclerosis status (carcinoma, etc. excluded) Organ Atherosclerosis status Aorta Atherosclerotic Nonatherosclerotic

Element cadmium 46 37

zinc

Cd :Zn

988 4 1,420e, 4

Bone Atherosclerotic Nonatherosclerotic Kidney Atherosclerotic Nonatherosclerotic

6.0 5.1 2,080 1,630

126g 113e 3,080 2,970

0.6803 • 4 0.553e, 4

Liver Atherosclerotic Nonatherosclerotic Lung Atherosclerotic Nonatherosclerotic

2873 1643

3,880 4,020

98 55

1,090 1,030

54 35

554 601

Skin Atherosclerotic Nonatherosclerotic

Mean levels of each element in each organ are presented by atherosclerosis status in table III. Cadmium levels were slightly higher in atherosclerotic than in nonatherosclerotic patients for all organs, although these differences were not significant statistically. Aorta and bone of the atherosclerotic patients differed in iron and zinc levels from those of the nonatherosclerotics.


1 Although all tests were run adjusting for age, race and sex, the means shown were adjusted only if at least one of these three had an effect significant at the 5-percent level. 2 Sample sizes ranged at 44-45 for atherosclerotics and 25-28 for nonatherosclerotics, except for lung tissue (with sample sizes 40 and 24, respectively). Decreased sample sizes are due to unavailability of specimens or to technical difficulties in the chemical analysis. 3 Difference between unadjusted means is significant at the 5-percent level. 4 Difference between means adjusted for age, race and sex is significant at the 5-percent level.


313

Fig. 2. Ratio of cadmium to zinc concentration in the kidney cortex, by age and by atherosclerosis status. Cases of cancer, scleroderma and acute pancreatitis are excluded. • = Atherosclerosis, O = nonatherosclerosis, ∆ = indefinite. Fig. 3. Ratio of cadmium to zinc concentration in the kidney cortex, by age and by blood pressure as indexed by cardiac weight. Cases of cancer, scleroderma, acute pancreatitis and valvular heart disease are excluded. • = Cardiac weight Z 500 g, O = cardiac weight < 500 g, ∆ = indefinite.


Such levels were lower in the aorta and higher in bone of atherosclerotic patients. After adjustment for age, race and sex, the statistical significance of these differences disappeared. In order to examine the association of Cd and Zn with atherosclerosis more closely, these elements were re-examined after exclusion of those cases whose diagnoses could detract from the relationship (if any) between cardiovascular disease and trace element level. Thus, all cancer cases (22 cases) were excluded, as well as a case of scleroderma, which disease is associated with a disturbed inorganic metabolism [LERICHE and JUNG, 1935; FELDMAN et al., 1969], and a case dying from acute pancreatitis. Table IV gives results for cadmium and zinc by atherosclerosis status. Statistical results are not unlike those for table III, but the difference in unadjusted cadmium liver levels is now significant, as is that for the adjusted aorta zinc levels. In order to test SCHRoEDER's hypothesis [1967 b] of a cardiovascular influence exerted by the cadmium-zinc ratio in the kidney, the association of the latter with atherosclerosis status was assessed. This association was found to be statistically significant (table IV). A scattergram of the cadmium-zinc ratio in the kidney by age and by atherosclerosis status is given in figure 2. The atherosclerotics seem to have a somewhat higher cadmium-zinc ratio in the middle age group. After further exclusion of 3 cases of valvular heart disease, the relation between hypertension (as indexed by cardiac weight) and cadmium-zinc in aorta and kidney was examined. The association between cardiac weight and aorta cadmium was positive and significant, both before and after adjustment for age, race and sex. The association between cardiac weight and aorta zinc was virtually zero both before and after adjustment for age, race and sex. The association of cardiac weight with kidney cadmium-zinc ratio was nonsignificant according to the F-test as used above, both before and after adjustment for age, race and sex. However, using a one-talled Student t-test,


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200 300 400 500 600 700 800 900 CARDIAC WEIGHT, G

the unadjusted ratio was significantly correlated with cardiac weight at the 5-percent level. A scattergram of the cadmium-zinc ratio in the kidney by age and by cardiac weight is given in figure 3. Persons with high cardiac weight at middle or old age tend to have increased kidney cadmium relative to zinc in this study. The relationship between this ratio and the cardiac weight is illustrated in figure 4 presented as a scattergram` while distinguishing between regions of residence. It appears that the correlation was due mainly to the residents of the Coastal Plain. Alternatively, when distinction was made in race instead of residence, it appeared that the correlation was due to blacks only.


Fig. 4. Ratio of cadmium zinc concentration in the kidney cortex, by blood pressure (as indexed by cardiac weight), and by region of last residence. Cases of cancer, scleroderma, acute pancreatitis and valvular heart disease are excluded. • = Piedmont, O = Coastal Plains, ∆ = other.

VGGRS/SHUMAN/GALLAGHER

316

The positive association between smoking and tissue cadmium confirms the suspicion voiced by FRIBERG et al. [1971] that cadmium intake via the respiratory route may make an important contribution to the total resorbed intake. It also confirms a similar correlation reported by LEWIS et al. [1972]. Bone and liver zinc, especially in the Coastal Plan, is low compared to results of other workers [TIPTON and Cοοκ, 1963 ; BOWEN, 1966]. It is uncertain whether these differences are real or due to methodologic variations. The higher cadmium levels in the Piedmont, on the other hand, are likely to be due to urban and industrial influences. The main kidney values for atherosclerotics and nonatherosclerotics with respect to the various trace elements are compared with the median values for decedents from heart disease and others, as found by ΡιPTOν [1960], in table V. Such a comparison was only possible for the elements cadmium, copper, lead and zinc. Our levels of lead were lower than those of Tιrroν and Cοοκ [1963]. We speculate that this difference is not due to methodology but may reflect lower pollution levels in North Carolina. In assessing the association between disease state and trace element level, the question arises what part is played by age, race and sex. If the latter three only determined the trace element level (by modifying cumulative intake and excretion) and not its relation with disease, then statistical adjustment for these three did not clarify the trace element-disease relationship, but confused it. If, on the other hand, age race or sex would not influence the trace element level, but merely modify its relationship with the disease state, then adjustment was warranted. It was found that in the large majority of cases the age effect alone accounted for the difference between adjusted and unadjusted test outcomes. The difference in aorta iron and zinc levels between atherosclerotics and nonatherosclerotics (table III, IV) is intriguing since it may signal increased susceptibility of the atherosclerotic arteries to noxious substances. The positive association between cardiac weight (indexing hypertension) and aorta cadmium level deserves further exploration with respect to possible causal mechanisms. In the kidney there is an association between cadmium-zinc ratio and presence of atherosclerosis. Likewise, there is a slight association between hypertension as indexed by cardiac weight and cadmium-zinc ratio. These relationships confirm the reports by others as mentioned earlier in this paper. Such findings do not exclude the possibility that increased cadmium-zinc


IV. Interpretation of Results


317

Table V. Means and sample sizes of element levels in four organs, by atherosclerosis status. Comparison with TτØκ's data [1960] Element

Means by pathology (from table ΙΙΙ)

Medians by cause of death [TYrroi, 1960]

Organ

atherononathero- heart sclerotic sclerotic (n=54-62) (n''39-43) (n=18)

accident

other

(n=87) (n=12)

Cadmium Aorta Kidney Liver Lung

43 2,180 264 911

39 1,790 176 532

< 50 3,000 158 50

< 50 2,800 180 50

< 50 4,600 240 < 50

Copper Aorta Kidney Liver Lung

151 281 472 3101

297 211 386 1352

60 250 610 135

95 260 510 120

87 280 770 135

Aorta Kidney Liver Lung

88 44 68 291

91 53 114 392

115 92 120 60

170 110 130 47

148 93 163 56

Aorta Kidney Liver Lung

1,040 3,210 4,100 1,0701

1,310 3,090 4,240 1,0232

1,500 4,600 3,300 1,150

1,950 4,500 3,700 1,300

2,300 5,500 4,500 1,400

Lead

Zinc

ratios are merely a manifestation of the hypertensive or atherosclerotic process. Alternatively, increased cadmium levels could merely indicate increased exposure to environmental atherogenic agents like smoked cigarettes. However, we tend to agree with Sc-1o030í that such findings strengthen the suspicion that environmental cadmium and zinc may explain some of the geographical differences in cardiovascular disease mortality. The finding that the association of the kidney cadmium-zinc ratio with the hypertension index


1 Sample sizes (n) range 45-48. 2 Sample sizes (n) range 30-32.


318

was found only for blacks suggests the possibility of hereditary molecular mechanisms and deserves further probing. Finally a word of caution. Even if the causal interpretation as projected above turns out to be correct, it is dangerous to extrapolate beyond the particular autopsy population investigated in North Carolina. This selective population differs in important respects from the general US population, and such differences have no doubt influenced their trace metal intake. V. Conclusion The nature of the environmental influence on cardiovascular disease has been investigated with respect to cadmium and zinc. Residents from a rural area with leached soils and high cardiovascular mortality were compared with residents from a more industrialized area. Regional differences in trace metal level fairly consistent among the various organs were found, but statistical significance was reached only by kidney zinc. Increased kidney levels of cadmium relative to zinc were found to be associated with atherosclerosis and somewhat associated with hypertension as indexed by cardiac weight. Therefore, to the extent that this association represents a causal effect, 50Ø00)01'S hypothesis [ 1967 b] concerning the nature of the environmental influence on cardiovascular disease was supported for this study population.

The peculiar geographic pattern of cardiovascular disease in nonmetropolitan US residents was explored in a high cardiovascular disease death area with leached soils, namely the North Carolina Coastal Plain. The South Atlantic Coastal Plan area is noted for its zinc deficiency and has a high stroke mortality rate. In the literature, zinc deficiency has been associated with impaired wound healing, and improvement in atherosclerotic patients after oral zinc administration has been reported. Competitive inhibition between zinc and cadmium actions at the molecular level has been demonstrated in experimental animals. Seven hospitals collaborated in furnishing tissues of long-term state residents. 33 lived in the Coastal Plain and 54 in the Piedmont, a control area, whereas 19 had lived elsewhere. Two aspects of cardiovascular disease were studied, namely atherosclerosis and hypertension. Atherosclerosis was asses-


Summary


319

sed by both clinical and pathological diagnosis : 58 decedents had atherosclerosis, 36 had not and 12 were equivocal. Hypertension was indexed by cardiac weight. For each autopsy, aorta, rib bone, kidney cortex, liver, lung and skin were analyzed by atomic absorption spectrometry for Cd, Cu, Fe, Pb and Zn. Epidemiologic interest concentrated on the organ-element combinations of conceptual relevance to cardiovascular disease. Aorta cadmium and zinc levels and kidney cadmium-zinc ratios were chief foci of attention. For nearly all organs examined, both cadmium and zinc had slightly higher levels in the Piedmont than in the Coastal Plain. Aorta and kidney zinc levels seemed low as compared to other US studies. Aorta cadmium levels and kidney cadmium-zinc ratios were somewhat higher in cardiovascular disease as compared to noncardiovascular disease patients for the combined areas. This result supports SCHROEDER's thesis [1967 b] that cadmium and zinc tissue levels may convey an environmental influence on cardiovascular disease status.

ΑνrΓscηκοw, N.: Experimental arteriosclerosis in animals; in COWDRY Arteriosclerosis, pp. 271-322 (Macmillan, New York 1933). AxELSS0N, Β.; DAHLGREN, S. Ε., and PτscATOR, Μ.: Renal lesions in the rabbit after long-term exposure to cadmium. Arch. environm. Hlth 17: 24-28 (1968). BARDER, Ε. W.: Die chronische Kadmiumvergiftung. Dtsch. med. Wschr. 76: 484-487 (1951). BERGMAN, Β.: The distribution and concentration of zinc and the effect of zinc deficiency in the mammalian body. Odont. Rev. 21: suppl. 20, pp. 3-54 (1970). BERLIN, Μ. ; HAMMARSTROM, L., and MAUNSBACH, A. B.: Micro-autoradiographic localization of water-soluble cadmium in mouse kidney. Acta radiat. ther. 2: 345-352 (1964). BORST, J. G. G. and BORST-DE GEUS, A.: Hypentension explained by Starling's theory of circulatory homoeostasis. Lancet i: 677-682 (1965). Bοwεν, H. J. Μ.: Trace elements in biochemistry (Academic Press, New York 1966). BRUINER, H. R.; LARAGH, J. H.; BAER, L.; NEWTON, Μ. A.; GOODWIN, F. T.; KRAKOFF, L. R.; BARD, R. H., and BURLER, F. R.: Essential hypertension. Renin and aldosterone, heart attack and stroke. New Engl. J. Med. 286: 441-449 (1972). CARROLL, R. E.: The relationship of cadmium in the air to cardiovascular disease death rates. J. amer. med. Ass. 198: 177-179 (1966). CRAWFORD, Μ. D. ; GARDNER, Μ. J., and MORRIS, J. N.: Cardiovascular disease and mineral content of drinking water. Brit. med. Bull. 27: 21-24 (1971). DAiL, L. K.: Salt and hypertension. Amer. J. clin. Nutr. 25: 231-244 (1972). DAvis, J. O.: What signals the kidney to release renin? Circulat. Res. 28: 301-306 (1971).


References

320

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322

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VOGRS/SHUMAN/GALLAGHER

323

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VGORS/SHUMAN/GALLAGREB

324

A. W.: Atherosclerotic heart disease and drinking water trace metals in North Carolina. Proc. 5th Ann. Conf. on Trace Substances in Environmental Health, 1971, pp. 525-534 (University of Missouri Press, Columbia 1972). WESTER, P. Ο.: Trace elements in heart tissue. Acta med. scand. 178: suppl. 439, pp. 1-47 (1965). WESTMORELAND, N.: Connective tissue alterations in zinc deficiency. Fed. Proc. 30: 1001-1010 (1971). WJLENS, S. L.: The effect of postural hypertension on the development of atheromatosis in rabbits fed cholesterol. Arch. Path. 19: 293-305 (1943). YouKTLis, E. J.; MARTZ, R. C.: HARRIS, P. D.; NIcoLL, P. A., and FoRNEY, R. Β.: Effects of cadmium on the minute blood vessels. Toxicol. appl. Pharmacol. 14: 628 (1969).

Author's address: A. W. VoοRS, MD, Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, Ν. C. (USA)


VOIRS,


325

Appendix

Sample of Partial Questionnaire used for Male Decedents North Carolina Health Study

1. Your name: 2. Your address •

(first)

(middle)

(maiden, if applicable)

(house number)

(street)

(town)

(county) (zip code)

(state)

(last)

(telephone number)

3. What is your relationship to the deceased? ❑ Wife ❑ Son ❑ Sister ❑ Brother ❑ Daughter ❑ Other, please specify • 4. List of all of his home addresses where he lived for more than 2 years beginning at birth, and also what the main source of drinking water was at this address. (Continue on reverse side if necessary.) Water source (check one) County

State

3 ~ C

v~i

~

~ ° α σ

., 4 n .ο ν

-' ~

~

>

G

a

ι,

ω

α: 3 Á


Town

Private well

Years (e.g., 1905-1920)

326


years

5. How many years did he spend in military service 9 6. What was his occupation for most of his adult life? (check one box) ❑ Farming, fishery, forestry and related occupations ❑ Clerical and sales ❑ Professional and managerial

❑ Bench work, machine trades and processing ❑ Construction work ❑ Other, specify:

Please briefly describe the main kind of work he did in this occupation:

7. a Was he frequently exposed to irritating smoke, dust, or fumes at his job? p Yes ❑ No b If yes, please list them•

8. For the last 5 years of his life, how much did he smoke of each of the following (check only one box per line): Type of tobacco

Yes, regularly No, never (5 or more per smoked day)

Yes, No, occasionally ex-smoker

Cigarettes Cigars Pipe a If he did smoke cigarettes, did he usually inhale?

❑ Yes

❑ No

Thank you for your cooperation.


b If he did smoke cigarettes, about how many per day did he usually smoke when he was smoking? ❑ Less than 1/2 pack per day ❑ About Ι % packs per day (1-5 cigarettes) (26-34 cigarettes) ❑ About 1/2 pack per day ❑ About 2 or more packs per day (35 or more cigarettes) (6-14 cigarettes) ❑ About 1 pack per day (15-25 cigarettes)

Water constituents and trace elements in relation to cardiovascular diseases.

Trace elements: an association with cardiovascular diseases and hypertension.

Mass balance of trace elements in Walker branch watershed: relation to coal-fired steam plants.

Trace elements in man.

Trace elements in animals.

Profile of Some Trace Elements in the Liver of Camels, Sheep, and Goats in the Sudan.

Atherosclerosis in relation to cholelithiasis and cholesterolosis.

Trace elements.

Trace elements.

Trace elements in diabetes mellitus.

Antioxidant status in blood of obese children: the relation between trace elements, paraoxonase, and arylesterase values.

Trace elements in the atmosphere.

Trace elements in Isoparorchis hypselobagri.

Urine level of trace elements in some adults in Bangladesh under a workplace environment.

Comparative study of trace elements in blood, scalp hair and nails of prostate cancer patients in relation to healthy donors.

Different behaviours in phytoremediation capacity of two heavy metal tolerant poplar clones in relation to iron and other trace elements.

Trace elements in soils and crops.

Minerals and trace elements in milk.

Trace elements and immune responses.

Trace Elements in Living Systems: From Beneficial to Toxic Effects.

Measurement of trace elements in urine.

Study on some trace elements content of certain internal organs and muscles of lambs.

Trace elements in intravenous feeding of infants.

Role of trace elements in senile cataract.